The present invention relates generally to the manipulation of genetic materials and more particularly to methods and materials for securing expression of exogenous, viral-vector-borne genes in mammalian cells.
Expression of exogenous genes has been attempted by employing a variety of host cell systems including bacterial cell, yeast cell and mammalian cell systems. Mammalian cell systems for exogenous gene expression have displayed distinct advantages when compared with both bacterial and yeast systems. In the lower organism cell systems, incomplete post-translational processing (i.e., glycosylation or proteolysis) of the exogenous gene may occur, resulting in loss of significant antigenic determinants and/or biological activity of the polypeptide coded for by the exogenous gene. By contrast, mammalian host cells provide the factors necessary for proper processing, proper secondary or tertiary structure and excretion or inclusion into the cell membrane. See, Berman, P., et al., Science, 222, 524-527 (1983).
A common protocol for expression of exogenous genes by mammalian cells maintained in culture is through infection of cells with viral vectors carrying exogenous gene DNA sequences. Typically, the exogenous gene is placed under the influence of viral controlling elements as a replacement for a deleted viral gene. A complementing, co-infecting helper virus is required to ensure propagation of the recombinant genome in the infected host cells by supplying the proteins ordinarily expressed by the deleted viral gene. As one example, SV40, a DNA tumor virus of the Papova virus group, which normally infects monkey kidney cells, has been employed extensively as a eukaryotic expression vector in this manner.
Another commonly-employed method for obtaining expression of exogenous genes in mammalian cells is through the introduction into the cells of shuttle vectors carrying the exogenous gene of choice. Shuttle vectors contain both bacterial plasmid sequences and viral DNA sequence, the former sequences allowing selection and replication in bacteria and the latter permitting expression and/or replication in mammalian cell culture. Therefore, like bacterial or yeast vectors, all manipulations involving the insertion of an exogenous gene and propagation of these vectors can be conveniently accomplished in E.coli prior to expression of the exogenous gene in mammalian cells. As one example, the above-described SV40 viral DNA has also been extensively employed in shuttle vector constructions.
An SV40-based viral vector may be constructed by replacing SV40 early gene regions or SV40 late gene regions with an exogenous gene sequence. If an exogenous gene is inserted to replace a deleted early viral gene DNA sequence coding for T antigen, the recombinant virus must be propagated in the presence of SV40 T antigen, e.g., supplied by simian COS-1 cells (ATCC CRL1650) or co-infection with a helper virus. Alternatively, if late viral gene DNA is excised from SV40 to permit insertion of the exogenous gene coding sequence, the early T antigen gene is present but the DNA sequences coding for expression of essential capsid proteins is absent. Therefore, these recombinant viruses must infect a host cell in concert with a "helper" virus which supplies the missing proteins. Early gene replacement viral vectors, which are easily propagated in COS cells which supply SV40 T antigen, are technically more adaptable to experimental manipulation than late gene replacement viral vectors, which require co-infection with a helper virus.
A disadvantage incurred in using the SV40 viral vectors for expression of exogenous genes in mammalian cells, resides in inherent limitations on the size of the viral vector. It has been concluded that the icosohedral symmetry of the SV40 virion imposes restrictions on the size of the DNA that could be encapsulated by its capsid proteins. Because the expression of the exogenous gene typically requires propagation of the recombinant molecules, the addition of exogenous genes without removal of viral sequences, or the insertion of genes larger than the viral sequences removed is precluded by the packaging constraints of SV40 [see, Liu, C., et al., "Expression of HE Surface Antigen Using Lytic and Non-Lytic SV40 Based Vectors in Eukaryotic Viral Vectors", Y. Gluzman, ed., Cold Spring Harbor Laboratory, Cold Spring, N.Y., 1982, pages 55-60; and Liu, et al., DNA, 1, pages 213-221 (1982)].
In Liu, DNA, 1, supra, an SV40 vector for the direct expression of exogenous genes was constructed by eliminating SV40 genome sequences between HindIII (1493) [6 nucleotides 5' to the initiation codon for the gene coding for the major SV40 late protein, VP1, which is essential in capsid formation] and BamHI (2533) [50 nucleotides 5' to the termination codon for that gene]. A unique EcoRI restriction endonuclease enzyme recognition site was introduced into the SV40 genome at the HindIII terminus to allow the SV40 fragment to be cloned into pBR322 and amplified. A BamHI/EcoRI exogenous gene sequence, e.g., HBsAg, is inserted into the SV40 fragment in place of the deleted VP1 sequence and the SV40-HBsAg fragment cloned into a pBR322 derivative and amplified. Cleavage with BamHI and self-ligation results in a recombinant virus plasmid vector, therefore, lacking only the coding region of VP1 and containing the whole protein coding region for T antigen. When the recombinant SV40/hepatitis B virus DNA was introduced into permissive monkey cells by DNA transfection in the presence of helper virus (tsA28), which supplies the capsid protein normally expressed by the deleted VP1, HBsAg was synthesized at a level comparable to that of VP1.
Liu, et al., also indicated that size restrictions exist with regard to the exogenous gene that could be inserted into this SV40 vector, approximately limited to the size of the coding region of VP-1 protein (about 900 base pairs).
SV40 recombinant virus vectors described in Gething, et al., "The Expression of the Influenza Virus Hemaglutinin Gene from SV40-HA Recombinants" in Eukaryotic Viral Vectors, Y. Gluzman, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1982, pages 29-33; and Gething, M., et al., Nature, 293, pages 620-625 (1981), provide for the insertion of an exogenous gene sequence coding for the influenza virus hemaglutinin (HA) gene to replace either the SV40 late or early gene coding regions.
In formation of a late replacement vector, the exogenous influenza virus hemaglutinin gene was inserted between the HpaII (346) and BamHI (2533) sites of the SV40 genome replacing the deleted late gene region. Thereafter, the recombinant viral genome SV40-HA was cloned into the BamHI site of an E.coli pBR322 derivative plasmid and propagated in E.coli. The recombinant SV40-HA genome was excised from the plasmid by BamHI digestion, purified and self-ligated to form the vector which contained the SV40 origin of DNA replication and an intact set of early genes including an intact copy of the gene coding for SV40 large T antigen. Presence of the early coding region and viral origin of replication permitted replication of the vector DNA in permissive simian cells and complementation by helper virus supplied SV40 capsid proteins for the assembly of infectious virions.
Similarly described in these references is an early replacement vector in which the HA gene was inserted into the SV40 genome between the HindIII (5171) and BamHI (2532) restriction endonuclease enzyme recognition sites. The recombinant viral genome was thereafter cloned, propagated, purified and ligated as described above to yield a vector containing the SV40 origin of DNA replication and an intact set of late genes. Because the vector lacked the early gene coding for large T antigen, it could not replicate in simian cells unless functional T antigen was supplied by using as a permissive host the COS-1 line of SV40 transformed monkey cells carrying an endogenously expressed copy of the T antigen gene. A low rate of productive infection of the COS-1 cells was observed.
"Shuttle vectors" are hybrid vectors containing bacterial plasmid sequences and portions of SV40 gene sequences allowing selection and replication in bacteria and transient expression and/or replication in mammalian cells. As a class, shuttle vectors cannot be maintained in mammalian cells where expression of an exogenous gene insert is lost after a few days. The shuttle vectors disclosed in Southern, et al., "Mammalian Cell Transformation with SV40 Hybrid Plasmid Vectors", in Eukaryotic Viral Vectors, Y. Gluzman, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1982), pages 41-45 include sequences from E.coli pBR322 that permit selection and propagation in E.coli and segments of the SV40 viral genome that constitute a defined eukaryotic transcription unit-promoter, coding region and polyadenylation site. The phosphotransferase gene from TN5 (neo) was subcloned into pBR322 and subsequently inserted into the pSV family of plasmid vectors. The resulting pSV-neo hybrid plasmids contained the pBR322 origin of DNA replication and the beta-lactamase selective marker gene, the neo gene segment from TN5, the SV40 origin of DNA replication, the SV40 early promoter, 5' to the neo gene, the SV40 DNA sequence of unspecified size at the 3' side of the neo gene including the small t antigen intervening sequence, and the SV40 early region polyadenylation signal. These plasmids also contain the entire T antigen coding region, resulting in a rather large vector, which cannot be converted to a lytic viral vector by simple deletion of the pBR322 DNA sequence. Further, these vectors could not be readily manipulated to allow replacement of the neo gene by a different gene of interest.
SV40 recombinant early replacement viral vectors have most recently been employed to express portions of the HBsAg according to a construction described by Laub, O., et al., J.Virol., 48, 271-280 (1983). The SV40-HBsAg recombinants were constructed by inserting the SV40 genome into the BamHI site of pBR322 for amplification. Partial digestion of the SV40 sequence with HindIII and thereafter BclI, allowed deletion of the early gene sequence between HindIII (5171) [8 nucleotides 5' to the translation initiation codon of the large T antigen gene] and BclI (2770) [77 nucleotides 5' to the termination codon of the large T antigen], resulting in recombinant pLSV. The exogenous HBsAg coding sequence was inserted into the pLSV recombinant genome as a TacI/BamHI fragment (and alternatively a AvaI blunt ended/BamHI fragment) for amplification in E.coli. Thereafter, digestion with BamHI removed the pBR322 portion of the recombinant and self-ligation resulted in plasmids containing an SV40 origin of replication, a functional set of SV40 late genes, HBsAG replacing the SV40 early genes, the SV40 early promoter sequence and polyadenylation site. Transfection of the vectors into COS cells which supply T antigen permitted propagation of the virus.
There has yet to be disclosed to the art an easily alterable intermediate SV40 recombinant shuttle vector which would allow ready insertion and replacement of a desired exogenous gene between an SV40 promoter and terminator in the same vector for expression in mammalian cells. The above-described SV40 recombinant vectors have intrinsic limitations on their use in mammalian cell systems for expression of exogenous genes, because for each desired exogenous gene, a new SV40 vector must be constructed. The absence of unique restriction endonuclease enzyme recognition sites both behind the SV40 early promoter and near the terminator prevents easy insertion of exogenous genes and requires the alteration of termini in both the SV40 genomes and the exogenous gene fragments as illustrated by the above-described prior art manipulations.
Further, evaluation of the efficacy of lytic early or late gene replacement viral vectors, which propagate as viruses when grown in COS cells or with a helper virus is procedurally complex and time-consuming in contrast to the rapid evaluation of a given vector construction permitted by use of shuttle vectors. Maintained in bacteria, shuttle vectors will transiently express an exogenous gene insert for several days upon introduction into mammalian cells. For the production of an exogenous gene product in large quantities, however, lytic viral vectors are considerably more efficient than shuttle vectors.
There exists, therefore, a need in the art for shuttle vectors which can be readily manipulated to express any desired exogenous gene by replacement of SV40 sequences under the control of SV40 promoters and terminators, and allows for the conversion of the shuttle vector into a lytic viral vector.